This invention relates to compounds that have utility as oligonucleotide intermediates, and to methods for making such compounds. The compounds are based on the 3-deazapurine core. The invention also generally relates to the field of "antisense" agents, agents that are capable of specific hybridization with a nucleotide sequence of an RNA. In particular, this invention relates to novel compounds that may be incorporated into oligonucleotides, these compounds include novel heterocyclic bases, nucleosides, and nucleotides. When incorporated into oligonucleotides, the 3-deazapurines of the invention can be useful for modulating the activity of RNA. Oligonucleotides are used for a variety of therapeutic and diagnostic purposes, such as treating diseases, regulating gene expression in experimental systems, assaying for RNA and for RNA products through the employment of antisense interactions with such RNA, diagnosing diseases, modulating the production of proteins, and cleaving RNA in site specific fashions.
It is well known that most of the bodily states in mammals including most disease states, are effected by proteins. Such proteins, either acting directly or through their enzymatic functions, contribute in major proportion to many diseases in animals and man. Classical therapeutics has generally focused upon interactions with such proteins in efforts to moderate their disease causing or disease potentiating functions. Recently, however, attempts have been made to moderate the actual production of such proteins by interactions with molecules that direct their synthesis, intracellular RNA. By interfering with the production of proteins, it has been hoped to effect therapeutic results with maximum effect and minimal side effects. It is the general object of such therapeutic approaches to interfere with or otherwise modulate gene expression leading to undesired protein formation.
One method for inhibiting specific gene expression is the use of oligonucleotides and oligonucleotide analogs as "antisense" agents. The oligonucleotides or oligonucleotide analogs complimentary to a specific, target, messenger RNA (mRNA) sequence are used. A number of workers have reported such attempts. Pertinent reviews include Stein, et al., Cancer Research, 1988, 48, 2659-2668; Walder, Genes & Development, 1988, 2, 502-504; Marcus-Sekura, Anal. Biochemistry, 1988, 172, 289-295; Zon, J. Protein Chemistry, 1987, 6, 131-145; Zon, Pharmaceutical Res., 1988, 5, 539-549; Van der Krol, et al., BioTechniques, 1988, 6, 958-973; and Loose-Mitchell, TIPS, 1988, 9, 45-47. Each of the foregoing provide background concerning general antisense theory and prior techniques.
Thus, antisense methodology has been directed to the complementary hybridization of relatively short oligonucleotides and oligonucleotide analogs to single-stranded mRNA or single-stranded DNA such that the normal, essential functions of these intracellular nucleic acids are disrupted. Hybridization is the sequence specific hydrogen bonding of oligonucleotides or oligonucleotide analogs to Watson-Crick base pairs of RNA or single-stranded DNA. Such base pairs are said to be complementary to one another.
Prior attempts at antisense therapy have provided oligonucleotides or oligonucleotide analogs that are designed to bind in a specific fashion to -- which are specifically hybridizable with -- a specific mRNA by hybridization. Such oligonucleotide and oligonucleotide analogs are intended to inhibit the activity of the selected mRNA -- to interfere with translation reactions by which proteins coded by the mRNA are produced -- by any of a number of mechanisms. The inhibition of the formation of the specific proteins that are coded for by the mRNA sequences interfered with have been hoped to lead to therapeutic benefits.
A number of chemical modifications have been introduced into antisense agents-- oligonucleotides and oligonucleotide analogs-- to increase their therapeutic activity. Such modifications are designed to increase cell penetration of the antisense agents, to stabilize the antisense agents from nucleases and other enzymes that degrade or interfere with their structure or activity in the body, to enhance the antisense agents' binding to targeted RNA, to provide a mode of disruption (terminating event) once the antisense agents are sequence-specifically bound to targeted RNA, and to improve the antisense agents' pharmacokinetic and pharmacodynamic properties. These modifications are designed to enhance the uptake of antisense agents-- oligonucleotides and oligonucleotide analogs -- and thus provide effective therapeutic, research reagent, or diagnostic uses.
Initially, only two mechanisms or terminating events have been thought to be operating in the antisense approach to therapeutics. These are the hybridization arrest mechanism and the cleavage of hybridized RNA by the cellular enzyme, ribonuclease H (RNase H). It is likely that additional "natural" events may be involved in the disruption of targeted RNA, however. These naturally occurring events are discussed by Cohen in Oligonucleotides: Antisense Inhibitors of Gene Expression, CRC Press, Inc., Boca Raton, FL (1989).
The first, hybridization arrest, denotes the terminating event in which the oligonucleotide inhibitor binds to the target nucleic acid and thus prevents, by simple steric hindrance, the binding of essential proteins, most often ribosomes, to the nucleic acid. Methyl phosphonate oligonucleotides, Miller, et al., Anti-Cancer Drug Design, 1987, 2, 117-128, and .alpha.-anomer oligonucleotides are the two most extensively studied antisense agents that are thought to disrupt nucleic acid function by hybridization arrest.
The second "natural" type of terminating event is the activation of RNase H by the heteroduplex formed between the DNA type oligonucleotide or oligonucleotide analog and the targeted RNA with subsequent cleavage of target RNA by the enzyme. The oligonucleotides or oligonucleotide analogs, which must be of the deoxyribose type, hybridize with the targeted RNA and this duplex activates the RNase H enzyme to cleave the RNA strand, thus destroying the normal function of the RNA. Phosphorothioate modified oligonucleotides are the most prominent example of antisense agents that are thought to operate by this type of antisense terminating event. Walder, et al., in Proceedings of the National Academy of Sciences of the U.S.A., 1988, 85, 5011-5015 and Stein, et al., in Nucleic Acids Research, 1988, 16, 3209-3221 describe the role that RNase H plays in the antisense approach.
To increase the potency via the "natural" termination events the most often used oligonucleotide modification is modification at the phosphorus atoms. An example of such modifications include methyl phosphonate oligonucleotides, where the phosphoryl oxygen of the phosphorodiester linking moiety is replaced or the nucleotide elements together are replaced, either in total or in part, by methyl groups. Other types of modifications to the phosphorus atom of the phosphate backbone of oligonucleotides include phosphorothioate oligonucleotides. The phosphorothioate modified oligonucleotides are thought to terminate RNA by activation of RNase H upon hybridization to RNA although hybridization arrest of RNA function may play some part in their activity. Backbone modifications are disclosed as set forth in U.S. patent applications assigned to a common assignee hereof, entitled "Backbone Modified Oligonucleotide Analogs," Ser. No. 703, 619 and "Heteroatomic Oligonucleotide Linkages," Ser. No. 903, 160, the disclosures of which are incorporated herein by reference to disclose more fully such modifications. Phosphoroamidites have been disclosed as set forth in an application having U.S. Ser. No. 918,362 and assigned to a common assignee hereof, entitled "Improved Process for Preparation of 2'-O-Alkylguanosines and Related Compounds," the disclosures of which are incorporated herein by reference to disclose more fully such modifications.
All applications of oligonucleotides and oligonucleotide analogs as antisense agents for therapeutic purposes, diagnostic purposes, and research reagents require that the oligonucleotides or oligonucleotide analogs be synthesized in large quantities, be transported across cell membranes or taken up by cells, appropriately hybridize to targeted RNA or DNA, and subsequently terminate or disrupt nucleic acid function. These critical functions depend on the initial stability of oligonucleotides and oligonucleotide analogs toward nuclease degradation.
A serious deficiency of unmodified oligonucleotides for these purposes, particularly antisense therapeutics, is the enzymatic degradation of the administered oligonucleotides by a variety of intracellular and extracellular ubiquitous nucleolytic enzymes, hereinafter referred to as "nucleases." It is unlikely that unmodified, "wild type," oligonucleotides will be useful therapeutic agents because they are rapidly degraded by nucleases. A primary focus of antisense research has been to modify oligonucleotides to render them resistant to such nucleases. These modifications have heretofore exclusively taken place on the sugar-phosphate backbone, particularly on the phosphorus atom. Phosphorothioates, methyl phosphonates, phosphoramidites, and phosphorotriesters (phosphate methylated DNA) have been reported to have various levels of resistance to nucleases. Backbone modifications are disclosed as set forth in U.S. patent applications assigned to a common assignee hereof, entitled "Backbone Modified Oligonucleotide Analogs," Ser. No. 703,619 and "Heteroatomic Oligonucleotide Linkages," Ser. No. 903,160, the disclosures of which are incorporated herein by reference to disclose more fully such modifications.
Other modifications to "wild type" oligonucleotides made to enhance resistance to nucleases, activate the RNase terminating event, and enhance the RNA-oligonucleotide duplex's hybridization properties include functionalizing the nucleoside's naturally occurring sugar. Sugar modifications are disclosed as set forth in PCT Application assigned to a common assignee hereof, entitled "Compositions and Methods for Detecting and Modulating RNA Activity and Gene Expression," PCT Patent Application Number PCT/US91/00243, International Publication Number WO 91/10671, the disclosures of which are incorporated herein by reference to disclose more fully such modifications.
Other synthetic terminating events, as compared to hybridization arrest and RNase H cleavage, have been studied in an attempt to increase the potency of oligonucleotides and oligonucleotide analogs for use in antisense diagnostics and therapeutics. Thus, an area of research has developed in which a second domain to the oligonucleotide, generally referred to as a pendant group, has been introduced.
The pendant group is not involved with the specific Watson-Crick hybridization of the oligonucleotide or oligonucleotide analog with the mRNA but is carried along by the oligonucleotide or oligonucleotide analog to serve as a reactive functionality. The pendant group is intended to interact with the mRNA in some manner more effectively to inhibit translation of the mRNA into protein. Such pendant groups have also been attached to molecules targeted to either single or double stranded DNA. Such pendant groups include, intercalating agents, cross-linkers, alkylating agents, or coordination complexes containing a metal ion with associated ligands. A discussion of pendant groups is set forth in PCT Application assigned to a common assignee hereof, entitled "Compositions and Methods for Detecting and Modulating RNA Activity and Gene Expression," PCT Patent Application Number PCT/US91/00243, International Publication Number WO 91/10671, the disclosures of which are incorporated herein by reference in order to disclose more fully such modifications.
Prior approaches using cross-linking agents, alkylating agents, and radical generating species as pendant groups on oligonucleotides for antisense diagnostics and therapeutics have several significant shortcomings. The sites of attachment of the pendant groups to oligonucleotides play an important, yet imperfectly known, part in the effectiveness of oligonucleotides for therapeutics and diagnostics. Prior workers have described most pendant groups as being attached to a phosphorus atom which affords oligonucleotide compositions with inferior hybridization properties. Prior attempts have been relatively insensitive, that is the reactive pendant groups have not been effectively delivered to sites on the messenger RNA molecules for alkylation or cleavage in an effective proportion. Moreover, even if the reactivity of such materials were perfect, (i.e., if each reactive functionality were to actually react with a messenger RNA molecule), the effect would be no better than stoichiometric. That is, only one mRNA molecule would be inactivated for each oligonucleotide molecule. It is also likely that the non-specific interactions of oligonucleotide compositions with molecules other then the target RNA, for example with other molecules that may be alkylated or which may react with radical species, as well as self-destruction, not only diminishes the diagnostic or therapeutic effect of the antisense treatment but also leads to undesired toxic reactions in the cell or in vitro. This is especially acute with the radical species that are believed to be able to diffuse beyond the locus of the specific hybridization to cause undesired damage to non-target materials, other cellular molecules, and cellular metabolites. This perceived lack of specificity and stoichiometric limit to the efficacy of such prior alkylating agent and radical generating-types of antisense oligonucleotide compositions is a significant drawback to their employment.
Reactive functionalities or pendant groups attached to oligonucleotide compositions previously described in the literature have been almost exclusively placed on a phosphorus atom, the 5-position of thymine, and the 7-position of purines. A phosphorus atom attachment site can allow a reactive group access to both the major and minor grooves. However, internal phosphorus modification results in greatly reduced heteroduplex stability. Attachments at the 3' and/or 5' ends are limiting in that only one or two functional groups can be accommodated in the oligonucleotide compositions. Such placement can interfere with Watson-Crick binding. Further, functionalities placed in the 5-position or 7-position of bases, pyrimidine and purine, respectively will reside in the major groove of the duplex and will not be in proximity to the RNA 2'-hydroxyl substrate. The 2' hydroxyl is the "trigger" point for RNA inactivation, and thus, any reactive functionalities must be in appropriate proximity to the receptive substrate located in the targeted RNA, especially the most sensitive point, the 2' -hydroxyl group.
Targeted RNA is inactivated by formation of covalent links between a modified oligonucleotide and the RNA 2' -hydroxyl group. A variety of structural studies such as X-ray diffraction, chemical reaction, and molecular modeling studies suggests that the 2' -hydroxyl group of RNA in a duplex or heteroduplex resides in the minor groove.
The half life of the perfectly formed duplex will be greatly effected by the positioning of the tethered functional group. Inappropriate positioning of functional groups, such as placement on the Watson/Crick base pair sites, would preclude duplex formation. Other attachment sites may allow sequence-specific binding but may be of such low stability that the reactive functionality will not have sufficient time to initiate RNA disruption.
Approaches to perfect complementation between modified oligonucleotides or oligonucleotides and targeted RNA will result in the most stable heteroduplexes. This is desired because the heteroduplex must have a sufficient half life to allow the reactive or non-reactive functionalities of this invention to initiate the cleavage or otherwise disruption of RNA function. The minor side or minor groove of the duplexes formed between such oligonucleotides or modified oligonucleotides and the targeted RNA has been found to be the greatly preferred site for functional group activity.
Therefore, functionalities placed on sequence-specific oligonucleotide compositions (via modified nucleosides) should preferably reside in the minor groove formed between the oligonucleotide composition and the targeted RNA, not interfere with duplex formation or stability, and initiate cleavage or disruption of the RNA. Accordingly, there remains a great need for antisense oligonucleotide compositions that are capable of improved specificity and effectiveness both in binding and in mRNA modulation or inactivation without the imposition of undesirable side effects.
It has now been found that certain positions on the nucleosides of double stranded nucleic acids are exposed in the minor groove and may be substituted without affecting Watson-Crick base-pairing or duplex stability. Reactive or non-reactive functionalities placed in these positions can best initiate cleavage and destruction of targeted RNA or interfere with its activity.
The functionalities point of attachment to the base units, which in turn may be converted to modified oligonucleotide, is critical in the design of compositions for sequence-specific destruction or modulation of targeted RNA. The functionalities must not interfere with Watson-Crick base pair hydrogen bonding rules, as this is the sequence-specific recognition/binding factor essential for selection of the desired RNA to be disrupted. Further, the functionalities should improve the oligonucleotides compositions' pharmacokinetic and/or pharmacodynamic properties, as well as the oligonucleotide compositions' transport properties across cellular membranes. The present invention addresses these, as well as other, needs by presenting novel oligonucleotide intermediates based on the core structure of 3-deazapurines.
The synthesis of the 3-deazaguanine core is known, Cook, et al., J. Am. Chem. Society 1975, 97, 2916; Cook et al., J. Med. Chem. 1978, 21, 1212. 3-deazaguanine is a potent guanine antimetabolite with significant antitumor, antiviral, antibacterial and antiparasitic activities. The corresponding nucleoside, 2' -deoxy-3-deazaguanosine, has exhibited a wide spectrum, Revankar, et al., J. Med. Chem., 27, 1389, 1984, of antiviral and antitumor activity in addition to antibacterial activity against E. coli, Burman, et al., Chem. Scripta 1986, 26, 15. Workers have made certain modifications to 3-deazaguanine (6-aminoimidazo [4,5-c]pyridine) and the corresponding nucleoside 2'-deoxy-3-deazaguanosine (6-amino-l-(2-deoxy-.beta.-D-erythro-pentofuranosyl))imidazo [4,5-c]pyridin-4(5H)-one resulting in a wide modulation of the heterocyclic ring system's biological activity. See, e.g., Hartman, et al., J. Labelled Compd. Radiopharm. 1985, 23, 35 (ring modifications); Revankar, et al., J. Med. Chem. 1984, 27, 1389 (peripheral modifications); Cook, et al., J. Org. Chem. 1978, 43, 289 (same); Revankar, supra (sugar modifications). Workers have attached certain tether functionalities to the 3-position of 3-deaza-adenine. The Chemistry of Heterocyclic Compounds, A. Weissberger, Ed., Imidazole and Derivatives, Part 1, Interscience, N.Y. (1953). However, there has been no investigation into the synthesis of 3-C substituted deazaguanine. The present invention is the first to set forth substitutions at the 3-C position of 3-deazaguanines and 3-deazapurine derivatives.
The bulkiest 3-C deazapurine substituent induces an unnatural 3'-endo/high-anti (-sc) conformation of the nucleoside. This preference for the anti-conformation may make the 2' -deoxy 3-deazapurines of the invention enhanced substrates for viral kineses. Further, substitutions at the C-3 aromatic carbon of the 3-deazapurine ring system are of interest because this influences the heterocycle's range of rotation about the glycosidic bond, Saenger, "Principles of Nucleic Acid Structure," Cantor C. R., Ed., Springer-Verlag, New York, 1983, potentially modifying biological activity, Saran, et al., Int. J. Quantum Chem. 1984 25, 743; Miles, et al., H.J. Theor. Biol. 1977, 67, 499. Further, lipophilic substituents at this position could change the transport efficiency of heterocyclic bases, heterocyclic base analogs, nucleosides, nucleoside analogs, nucleotides, nucleotide analogs, and oligonucleotides compositions.